title: Integrity of ATP binding site is essential for effective inhibition of the intrinsic apoptosis pathway by NAIP.
abstract: The importance of the ATP binding site of human Neuronal Apoptosis Inhibitory Protein (NAIP) on its ability in prevention of intrinsic apoptotic pathway was investigated. Thus, ATP binding lysine 476 of NAIP, which is located at the Nucleotide Binding Oligomerization Domain (NOD) was mutated to threonine and the effect of this mutation on autoproteolysis of procaspase-9 and the cleavage of procaspase-3 by apoptosome was investigated. Formation of apoptosome was induced by the addition of cytochrome c and dATP to lysates of HeLa cells transfected with pcDNA-NAIP or pcDNA-NAIP (K476T). Full length wild type NAIP prevented the cleavage of both procaspase-9 to caspase-9 and procaspase-3 to caspase-3. However, K476T variant of NAIP did not block autocleavage of procaspase-9 efficiently. Furthermore, cleavage pattern of procaspase-9 was altered in the presence of mutant NAIP. Interestingly no effect on the procaspase-3 cleavage by apoptosome was observed. The presence of NOD domain by itself had no effect on autocleavage of procaspase-9 yet slightly reduced the cleavage of procaspase-3 by apoptosome. Pull down experiment showed direct interaction of the NOD domain of NAIP with the CARD-NOD domain of Apoptotic Protease Activating Factor 1 (APAF-1). The physical association of these domains was confirmed by pull-down assays. These observations taken with previous findings indicate that the integrity of the NOD domain is essential for effective inhibition of procaspase-9 and procaspase-3 cleavage by the NAIP protein.
authors.affiliation: Department of Pharmacology and Toxicology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran 1417653761, Iran.
authors.forename: Sarvenaz
authors.initials: S
authors.lastname: Karimpour
authors.forename: Jamshid
authors.initials: J
authors.lastname: Davoodi
authors.forename: Mohammad-Hossein
authors.initials: MH
authors.lastname: Ghahremani
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D017209:Apoptosis / Q000235:genetics*; D053478:Apoptosomes / Q000037:antagonists & inhibitors* / Q000378:metabolism; D053477:Apoptotic Protease-Activating Factor 1 / Q000378:metabolism; D001665:Binding Sites; D053148:Caspase 3 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D006801:Humans; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D051616:Neuronal Apoptosis-Inhibitory Protein / Q000235:genetics* / Q000378:metabolism*
chemical_list: C506592:APAF1 protein, human; D053478:Apoptosomes; D053477:Apoptotic Protease-Activating Factor 1; D051616:Neuronal Apoptosis-Inhibitory Protein; D000255:Adenosine Triphosphate; D053148:Caspase 3; D053453:Caspase 9; D008239:Lysine